RNAi Delivery to Vascular Endothelium Increasingly Validated

Less than a month after Napoleone Ferrara from Genentech was recognized with a 2010 Lasker Prize for identifying VEGF as the central actor in blood vessel formation, a prize widely regarded as the stepping stone towards the Nobel Prize in Physiology or Medicine, a press release by Alnylam seems to suggest that RNAi delivery to the vascular endothelium is appropriately reaching critical mass. Before that, the most important body of work in this area probably came from Silence Therapeutics, which, despite its apparent quality, got me a bit worried given what I perceived as a certain lack of enthusiasm in the commercial RNAi Therapeutics space and, more importantly, third party scientific validation.

It is particularly exciting that the silencing of endothelial gene markers following a single dose persisted for two months. Since the duration of silencing is critically dependent on the cell type, for example its proliferation rate, this bodes very well for all delivery technologies targeting the vascular endothelium.

Alnylam mentions that the new LNPs (liposomal nanoparticles) that work for endothelial siRNA delivery stem from their collaboration with the MIT which as we know has involved positively charged ‘lipidoids’. Seen in light of the data by Silence (cationic lipid-siRNA)/Intradigm (RGD-targeted PEI polymers), the picture that is emerging is that LNPs that comprise positively charged lipids have a natural propensity of being taken up by vascular endothelial cells. Although I haven’t seen the details yet, it is to be expected that a number of parameters beyond positive charge, such as the method of formulating the particles, e.g. SNALP-like siRNA encapsulation versus Atuplex-like lipoplex formation, determine the efficiency of the functional uptake of these particles.

Beyond Alnylam and Silence Therapeutics, which with Atu-027 already has an endothelial cell-targeting RNAi Therapeutics in the clinic, Tekmira should also have considerable expertise in this area. Similar to optimizing LNP delivery to the liver, leadership in endothelial RNAi will depend on first empirically determining the structure-function relationships of these particles and then the biological pathway by which the uptake occurs. To my knowledge, it is still unclear whether receptor-mediated uptake, non-specific macropinocytosis ('cell drinking'), or the limited capacity of endothelial cells for phagocytosis is involved. This will also be important to understand as more targeted technologies will be developed.

One trade-off that current technologies might suffer from is that their positive charge at physiological pH may cause them to be slightly more toxic compared to negatively or neutrally charged formulations. This may also tie in with Phil Haworth’s comments in my last interview with him that Silence Therapeutics will initially focus on acute indications with high unmet needs. But again, everything is toxic at sufficiently high concentrations and it is encouraging that Atu-027 is still in its dose-escalating phase according to the last clinical update by the company.

Progress also reported for systemic RNAi delivery to immune cells

Following up on their declared pursuit of vaccine opportunities, Alnylam also highlighted progress in the systemic delivery of siRNAs to immune cells. Previously, LNPs comprising lipids derived from the KC2 series of next-generation ionizable lipids had been described to silence the immune cell marker CD45 with an ED50 of approximately 1mg/kg following intravenous administration (see RNAi delivery roundtable). The new report suggests some improvement over those formulations, stating that a 95% knockdown of CD45 was achieved, with an ED50 of as little as 0.2mg/kg. It will be very interesting to see the scientific details and discussions on these experiments (note: this last paragraph was corrected from an earlier version that mistakenly stated that the 95% knockdown was achieved with 0.2mg/kg).

Uptake of RNAi triggers by immune cells per se is not entirely new. It is a case of turning lemons into lemonades, as the non-specific uptake of nanoparticles by phagocytic cells has been long lamented. However, these cells play a central role in at least as many important diseases as endothelial cells do- so why not harness the efficient uptake of particulates in those cells for therapeutic purposes? Sure, being taken up and being functionally released into the cytoplasm are 2 separate issues, but Tekmira’s Ebola work has shown already that LNPs can trigger gene silencing in macrophages. Again, it will be important to delineate the functional uptake pathways and to employ chemistry to increase the efficiency with which LNPs can harness them.

Once thought of as liver-only formulations, LNPs are showing more and more their considerable versatility. I remember well a talk given by Tekmira’s CSO Ian MacLachlan at Stanford two years ago when he demonstrated that by even only slightly changing the formulation parameters, strikingly different patterns of biodistribution can be achieved. Now it ‘only’ takes one of the LNP/SNALP programs in the clinic to show some efficacy, and the pharmaceutical industry to change its approach towards drug target selection, for the liposome to finally shed its image as the unloved, but necessary stepchild of the industry.